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Changes in In Vitro protein-synthesizing activity of embryonic fowl liver

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Title:
Changes in In Vitro protein-synthesizing activity of embryonic fowl liver
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Chu, Mon-Li Hsiung, 1948-
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English
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viii, 101 leaves. : ill. ; 28 cm.

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Subjects / Keywords:
Albumins ( jstor )
Amino acids ( jstor )
Embryos ( jstor )
Gels ( jstor )
Liver ( jstor )
pH ( jstor )
Polyribosomes ( jstor )
Protein synthesis ( jstor )
Ribosomes ( jstor )
RNA ( jstor )
Liver ( lcsh )
Proteins -- Synthesis ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 96-100.
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Manuscript copy.
General Note:
Vita.
Statement of Responsibility:
Mon-li Hsiung Chu.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Full Text
CHANGES IN IN VITRO PROTEIN-SYNTHESIZING ACTIVITY
OF EMBRYONIC FOWL LIVER
By
MON-LI HSIUNG CHU
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGEMENTS
The author wishes to express her deep appreciation and gratitude
to her research director, Professor Melvin Fried, for his guidance and
encouragement during the execution of this work.
The author also wishes to express her appreciation to her super
visory committee members, Dr. R. P. Boyce, Dr. R. J. Mans, and Dr. C.
Moscovici, for their suggestions and criticism.
Special thanks are due to Dr. H. M. Jernigan whose helpful
suggestions contributed much to this work and to Mrs. S. Eoff for
performing amino acid analyses.
A very special thanks is also expressed by the author to her
parents, who have made her education possible, and to her husband,
whose understanding and constant encouragement made this work easier.


TABLE OF CONTENTS
Page
Acknowledgements
List of Tables iv
List of Figures v
Abstract
Introduction 1
Mechanism and Control of Protein Synthesis
Liver Protein Synthesis 6
Research Objectives 11
Materials and Methods 13
Materials 13
Biological Preparations 14
Biochemical Determinations 22
Results and Discussion 30
Characteristics of the Cell-Free Protein-Synthesizing
System Derived From Embryo Livers at Various
Stages of Development 30
Distribution and Activity of Free and Membrane-Bound
Polysomes 62
Albumin Synthesis 79
Conclusions
Bibliography 96
Biographical Sketch 101
iii


LIST OF TABLES
Table Page
1 Dependence of protein synthesis on various components
of the reaction mixture
2 Subcellular distribution of Ami noacyl-tRNA
synthetase activity .
3 Amino acid requirement of the pyrophosphate-ATP rr
exchange reaction
4 Ribonuclease activity in liver subcellular
fractions derived from embryos of various
h Q
ages
5 Ribonuclease inhibitor activity in cell sap derived
from embryos of various ages 61
6 Distribution of free and membrane-bound polysomes
in homogenates of 10-day and 17-day mixture 66
7 Amino acid incorporating activity of free and
membrane-bound polysomes before and after Triton
X-I00 treatment 68
8 Ribonuclease activity in free and membrane-bound
polysomes 77
9 Albumin synthesis by membrane-bound and free liver
polysomes derived from embryos of various ages. 87
10Albumin synthesis by 3000 X g supernatant derived
from embryos of various ages 92


LIST OF FIGURES
Figure
Page
1
Preparation of total and salt-washed polysomes
15
2
Preparation of total cytoplasmic and free
polysomes from postnuclear supernatant
17
3
Preparation of free and membrane-bound polysomes
from postmitochondrial supernatant
19
4
3
Effect of cell sap concentration on the [ H] lysine
incorporation into protein
33
5
3
Effect of polysome concentration on the [ H] lysine
incorporation into protein
34
6
3
Time course of [ H] lysine incorporation into protein . .
35
7
Amino acid incorporating activity of cell-free systems
derived from 12-day and 19-day embryos
36
8
Amino acid incorporation by total polysomes derived
from embryos at various stages of development
38
9
Sucrose gradient profiles of total polysomes
derived from various age embryos
40
10
Free amino acid content of the cell sap derived
from embryos at various stages of developement
43
11
Lysine profiles from amino acid analysis
44
12
Amino acid incorporating activity of cell saps
derived from embryos of various ages
46
13
3
Cell sap dependence of [ H] lysine incorporation
47
14
3
Time course of incorporation of [ H] lysine from
[3|i] lysyl-tRNA into polypeptide
49
15
Effect of cell sap concentration on the incorporation
of [3H] lysyl-tRNA into polypeptide
50
16
0
Lysine incorporation from [ HJ lysyl-tRNA into polypeptide
by cell saps derived from embryos of various ages
51
17
Time course of pyrophosphate-ATP exchance assay
55
v


18 Effect of cell sap concentration on the pyrophosphate
exchange reaction 56
19 Ami noacyl-tRNA synthetase activity of cell sap
derived from embryos of various ages 58
20 Distribution of free and membrane-bound
polysomes in livers at various stages of
development 64
21 Sucrose gradient profiles of membrane-bound and
free polysomes prepared from 19-day embryonic
livers without using detergent 70
22 Time courses of amino acid incorporation of
membrane-bound and free polysomes 72
23 Sucrose gradient profiles of membrane-bound
polysomes after Triton X-100 treatment 74
24 Sucrose gradient profiles of free polysomes
after Triton X-100 treatment 76
25 Polyacrylamide gel electrophoresis of cell-free
protein products released from membrane-bound
and free polysomes before sonication ...... 81
26 Polyacrylamide gel electrophoresis of cell-free
reaction products released from membrane-bound
and free polysomes by sonication 83
27 Polyacrylamide gel electrophoresis of cell-free
protein products of 16-day membrane-bound
polysomes remaining after precipitation with
antiserum against albumin ... 8c
28 SDS-polyacrylamide gel electrophoresis of the cell-
free protein products precipitated with antiserum
against albumin 85
29 Polyacrylamide gel electrophoresis of cell-free protein
products synthesized by 3,000 X g supernatant from
livers at various stages of development 90
vi


Abstract of Dissertation Presented to t'ne Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
CHANGES IN IN VITRO PROTEIN-SYNTHESIZING ACTIVITY
OF EMBRYONIC FOWL LIVER
By
Mon-Li Hsiung Chu
March, 1975
Chairman: Melvin Fried
Major Department: Biochemistry
At selected times during chick embryo liver development, a
cell-free protein synthesizing system composed of polysomes and cell
sap was prepared. The amino acid incorporating activity of cell sap
decreased with increasing developmental age when assayed using
standard polysomes. Polysomes prepared from embryos of different
ages were uniformly active in amino acid incorporation when assayed
with standard cell sap. Cell sap ami noacyl-tRNA synthetase activity
remained constant during development, whereas the activity in trans
ferring amino acid from ami noacyl-tRNA into polypeptide decreased with
developmental age. Ribonuclease activity in the embryonic liver in
creased rapidly with age. The decline in amino acid incorporating
activity of the cell sap is ascribed to the lack of a cell sap component
involved in a stage of protein synthesis subsequent to the amino-
acylation of the tRNAs or to increasing ribonuclease activity, or both.
As development progressed, the free polysome content of embryonic
liver decreased, while membrane-bound polysome content increased markedly.
Total polysome content remained relatively constant during development.
v 1 i


The increase in membrane-bound polysome content correlates with and
may be responsible for the increase in the secretion of serum
proteins by liver cells during development.. Membrane-bound poly
somes were less active in amino acid incorporation than free poly
somes derived from embryos of the same age and this activity difference
was more pronounced in older embryos. The activity of free polysomes
remained constant with age. High ribonuclease activity was found in
the membrane-bound polysome preparation. The difference in amino acid
incorporating activity and ribonuclease activity between free and
membrane-bound polysomes was abolished after polysomes were treated
with Triton X-100. Ribonuclease activity present in the membrane,
therefore, probably inhibits the amino acid incorporating activity of
membrane-bound polysomes accumulating upon maturation of the embryo.
Albumin synthesis in the cell-free system was detected by poly
acrylamide gel electrophoresis and immunoprecipitation. Membrane-
bound polysomes were found to be the major site for albumin synthesis
in embryonic liver cells. The percentage of albumin in total protein
synthesized by membrane-bound polysomes derived from embryos of different
ages was relatively constant. Measurement of total protein synthesis
by the 3,000 X g supernatant of liver homogenate showed that the
percentage of albumin synthesis increased in parallel with develop
mental age. Correlations between these results and the differentiation
of serum proteins are discussed.
vm


INTRODUCTION
Mechanism and Control of Protein Synthesis
Spectacular progress in our knowledge of the biochemistry of
protein synthesis has occurred in the past two decades. Much of our
present understanding of the mechanism and control of protein synthesis
has been obtained from studies in cell-free systems. The first such
system was prepared from rat liver (Siekevitz and Zamecnik, 1951;
Siekevitz, 1952; Zamecnik and Keller, 1954). Since then, cell-free
protein-synthesizing systems have been prepared from a wide range of
different organisms. Among them are the rabbit reticulocyte system,
first studied by Schweet et al. (1958), and the E. coli system, first
studied by Lamborg arid Zamecnik (1960). From studies on these systems,
it is now known that protein synthesis takes place on ribosomes; that
enzymes and factors present in the high speed supernatant of cell
homogenate (cell sap) are required for protein synthesis; that messenger
RNA (mRNA) provides the code for amino acid sequence of each protein;
that ribosomes move along the mRNA as protein synthesis proceeds. Since
several ribosomes can be accommodated on a single mRNA molecule, poly
ribosomes (polysomes) are formed. The presence of polysomes indicates
that cells are actively synthesizing proteins. The events involved in
protein synthesis may be divided into four stages: activation of amino
acids, polypeptide chain initiation, chain elongation and chain termi
nation.
1


2
The first step in protein synthesis is the ATP-dependent activation
of amino acids by a class of enzymes known as ami noacyl-tRNA synthetases,
each of which is specific for one amino acid (Hoagland, 1955; Hoagland,
Keller and Zamecnik, 1956). The activation reaction occurs in two sep
arate steps with the formation of ami noacyl-adenylate as an intermediate
(Hoagland et at. 1957).
,, ++
Mg
.Amino acid+ ATP+ Synthetase ~ ^ (aminoacyl AMP + Synthetase) + PPi (1)
Mg+*
(Ami noacyl AMP + Synthetase) + tRNA Ami noacyl tRNA+ AMP + Synthetase (2)
Synthetase
Amino acid + tRNA<- - Ami noacyl tRNA + AMP + PPi (3)
The activation reaction is usually assayed by measuring the amino acid-
32
dependent rate at which P labeled pyrophosphate is incorporated into
added ATP in Reaction (1) catalyzed by the synthetases (Berg, 1956).
Initiation of protein synthesis begins with the binding of
initiating ami noacyl-tRNA and mRNA to the small subunit of the ribosome
in the presence of GTP and initiation factors to form the "initiation
complex," which then binds to the large subunit of the ribosome to form
a complete ribosome (Haselkorn and Rothman-Denes, 1973). Initiation
factors can extracted from the small ribosome subunit with 0.5 to
2.0 M NH^Cl KOI (Shafritz et al. > 1970; Heywood and Thompson, 1971;
Kaempfer and Kaufman, 1972).
Elongation of polypeptide chain can be considered as a three-step
process (Haselkorn and Rothman-Denes, 1973). The first step is the bind
ing of the incoming ami noacyl-tRNA to the acceptor site (A site) of the
ribosome. This binding requires GTP and an elongation factor termed
EF-T in prokaryotes and EF-1 in eukaryotes. The second phase is the


3
formation of a peptide bond linking the amino group of the ami noacyl-tRNA
with the peptide moiety of the peptidyl-tRNA, located at the peptidyl
site (P site) of the ribosome, to form a new peptidyl-tRNA, one amino
acid unit longer. This step is catalyzed by the enzyme, peptidyl
transferase, which is an integral part of the large ribosome subunit
(Monro, 1967). The third step is the translocation of the new peptidyl-
tRNA from.the A site to the P site of the ribosome, thus freeing the
A site for the next ami noacyl-tRNA (Traut and Monro, 1964). The trans
location process requires GTP and another elongation factor, termed EF-G
in prokaryotes and EF-2 in eukaryotes.
Polypeptide chain termination is signaled by termination codons
(UAA, UAG and UGA) in the mRNA. When a termination codon is reached, the
ribosome binds a release factor, R-| or R^, which activates peptidyl trans
ferase, which then hydrolyzes the ester linkage between polypeptide and
tRNA (Caskey et at. 1969; Capecchi and Klein, 1969).
Changes in the protein synthetic capacity of cells have been reported
to occur in response to altered nutritional conditions, hormone stimulation
and various pathological states (Pain and Clemens, 1973). These alterations
of protein synthetic activity are often ascribed to variations in ribosomal
and/or supernatant activities.
Several mechanisms could operate to alter the protein synthetic
activity of ribosomes. The rate of protein synthesis depends on the
number of ribosomes per cell, the proportion of ribosomes associated
with mRNA and the protein-synthesizing activity of polysomes (Monro
et al., 1953; Wilson and Hoagland, 1967).
Current evidence suggests that chain initiation is the rate
limiting step for translation in most cells. The number of active


4
ribosomes found in polysomes is regulated by the initiation process
(Kaempfer, 1971). Messenger-specific initiation factors, capable of
discriminating between classes of mRNAs, have been implicated in the
regulation of protein synthesis in eukaryotes. Heywood (1970)
demonstrated that initiation factors removed from chick muscle ribo
somes by a high salt wash were required for both the binding of muscle
mRNA to ribosomes and the mRNA-directed synthesis of myosin on reticulo
cyte ribosomes. Ilan and lian (1971) reported that, during insect
development, there was a stage-specific initiation factor which promoted
the formation of the complete initiation complex only with mRNA extracted
from the same stage of development. However, other workers have found no
requirement for specific factors for translation of mRNA in heterologous
system (Lane et al., 1971; Rhoads et al., 1971; Sampson et al., 1972).
Elongation factors, EF-1 and EF-2, are present both free in cell
sap and bound to ribosomes. Those factors, when bound to ribosomes, may
influence the activity of ribosomes in a cell-free system. Alexis et al:
(1972) found that crude muscle ribosomes from protein-deficient rats showed
lower protein-synthesizing activity than ribosomes from normal rats. The
activity difference between these two kinds of muscle ribosomes were
related to a difference in the content and/or activity of non-ribosomal
factors associated with the ribosome preparations rather than to alter
ations in the ribosomes themselves. An increase in EF-1 activity in
the spleen of rats was observed following immunization (Willis and Starr,
1971). In HeLa cells, EF-2 content increased during rapid growth and
decreased when growth slowed (Hendriksen and Sami son, 1972). In addition,
much of the EF-2 was present in the cell sap when the growth rate was high,


5
while a large proportion of EF-2 was associated with 80 S ribosomes
when protein synthesis was restricted.
Ami noacyl-tRNA synthetases regulate the rate of translation in
several different ways. First, the activity of the synthetases varies
with the rate of general protein synthesis in various physiological states.
Diabetes, which lowers the rate of protein synthesis in rat muscle, also
lowers the activity of synthetases in muscle cell sap (Pain, 1971).
Secondly, changes in the pattern of protein synthesis during growth
and development are often accompanied by changes in the spectrum of
activity of synthetases specific for different amino acids. The
synthesis of phosvitin, a serine-rich egg yolk protein, by the liver
of laying hens was accompanied by an increase in seryl-tRNA synthetase
level (Beck, Hentzen and Ebel, 1970). Thirdly, during protein or amino
acid deficiency, synthetase activity rises, facilitating amino acid con
version into protein. One example of such a phenomenon is that protein
deficiency in the rat results in increased activity of hepatic aminoacyl-
tRNA synthetases (Mariani, Spadoni and Tomassi, 1963).
The level of ribonuclease and ribonuclease inhibitor activities
has been shown to vary in different metabolic states of the cell. Kraft
and Shortman (1970) suggested that high inhibitor/ribonuclease ratios
were associated with the state of cytoplasmic RNA accumulation while
low ratios were associated with net RNA catabolism. The level of
cellular RNA, especially that of mRNA, controls the rate of protein
synthesis. A decrease in ribonuclease activity (Arora and de Lamirande,
1967) and an increase in ribonuclease inhibitor activity (Shortman,
1962; Moriyama et al. 1969) have been noted in rats following partial
hepatectomy. Protein deficiency in the rat resulted in lower ribo-


6
nuclease inhibitor activity and higher ribonuclease activity in the
liver cell as compared with well-fed control rats (Sheppard et al.,
1970). The low amino acid-incorporating activity of cell-free systems
derived from chicken liver was ascribed to an uninhibited ribonuclease
in the chicken liver cell sap (Siler and Fried, 1968). Burka (1970)
found a ribonuclease activity in the reticulocyte cell-free system
which was operating during the period when protein synthesis occurred
but was not active at 0C.
Liver Protein Synthesis
The liver's special function of secreting considerable amounts of
proteins into blood plasma makes this tissue particularly attractive
to those interested in studying the mechanism of regulation of protein
synthesis. In addition, the liver is a large discrete organ and is
relatively easy to fractionate into subcellular components. Albumin is
the most abundant of all the proteins found in the serum of most
vertebrates, representing 40-50% of the total plasma proteins (Engle
and Woods, 1960). Some of the early studies on albumin synthesis were
carried out in chicken liver. Peters and Anfinsen (1950) demonstrated
a net synthesis of serum albumin when slices of chicken liver were in
cubated with ^rC0,p. Peters (1959) studied the intracellular distribution
of the newly synthesized albumin. He found that if the chicken liver
slices were disrupted and fractionated, some albumin was released from
both mitochondrial and microsomal fractions by deoxycholate treatment.
Of these two fractions, microsomal albumin was found to be more radio
active. He tested the effectiveness of various reagents in releasing


7
serum albumin from the microsomal pellet and found that only those
substances which dissolved lipid were effective in releasing serum
albumin. Peters concluded that albumin was held in intimate associ
ation with the lipid membrane. He also characterized the microsomal
albumin with regard to sedimentation rate, electrophoretic mobility
and N-terminal and C-terminal amino acid residues (Peters et dl. 1958).
Continuation of such studies in the cell-free system was unsuccessful,
because the microsome fraction isolated from chicken liver was
relatively inactive in amino acid incorporation as compared with that
from rat liver (Campbell and Kernot, 1959). Therefore, the rat liver
has been used as a model system for studying albumin synthesis by most
investigators.
In protein-secreting cells such as liver cells, some polysomes
are bound to the membranes of the endoplasmic reticulum and some poly
somes are free in the cytoplasm (Palade and Siekevitz, 1956). Membrane-
bound polysomes are thought to be engaged in making those proteins
secreted by the cell, whereas free polysomes synthesize proteins that
are to be retained within the cell (Siekevitz and Palade, 1960; Vassalli,
1967). Redman (1968) first demonstrated that membrane-bound polysomes
were the main or exclusive site of albumin synthesis in rat liver cells.
Takagi and Ogata (1968) made similar findings. Hicks et at. (1969) com
pared the efficiency of free polysomes and total (free and membrane-bound)
polysomes of rat liver cells in making ferritin, a retained protein,
and albumin in a cell-free system. They showed that the total polysome
fraction was more efficient for albumin synthesis, presumably because it
contained membrane-bound polysomes. On the other hand, free polysomes


8
viere more efficient in ferritin synthesis. Uenoyarna and Ono (1972)
reported that albumin was synthesized by free polysomes from 5123
hepatoma, a system in which the synthesized albumin is not secreted
but retained in the cell. Taylor and Schimke (1973) demonstrated
that rat liver polysoma1 RNA was capable of directing albumin synthesis
in a cell-free system derived from rabbit reticulocytes. The RNA
extracted from membrane-bound and free polysomes of rabbit liver
has been shown to direct albumin and ferritin synthesis in a reticulo
cyte cell-free system (Shafritz et al., 1973; Shafritz, 1974a).
Albumin synthesis during embryonic development is an interesting
subject for studying the control of protein synthesis. The chick
embryo is an ideal organism for such study, because it is relatively
easy to obtain in large quantities and the embryonic blood is separate
from the maternal circulation. The blood proteins of the chick embryo,
especially in early stages of development, are very different from the
pattern characteristic of adult chicken. There is an increase in the
number of serum proteins as well as an increase in the concentration of
most components with age (Weller and Schechtman, 1962). Albumin first
appears as a distinct component in 9-day serum, its concentration
increasing from 13% of total serum proteins at the 9th day of develop
ment up to 50% at the 19th day and then showing a slight decrease by
hatching time on the 21st day. The differentiation of serum proteins
from embryonic to adult type is probably a direct result of functional
differentiation of the liver cells in their increasing ability to
synthesize adult proteins.
The chick liver appears at the end of the 2nd day of embryonic
life. It weighs approximately 2 mg on the 5th day, 50 mg on the 10th


9
day and 1 g at hatching time (Romanoff, 1967, p. 68). The growth of
the liver, expressed as a percentage of its hatching weight, when
plotted against incubation time, gives a characteristic sigmoid-shaped
curve. However, when the growth rate of liver is plotted against time,
there is a rapid fall from very high to low values between the 7th and
9th day of incubation. Thereafter, the growth rate decreases gradually
until hatching time (Romanoff, 1967, p. 267). It is believed that the
decrease in the specific rate of growth is due to the slowing down of
metabolism caused by the reduction in the rate at which the tissues are
supplied with nutrients (Byerly, 1932). Early embryonic liver cells
lack many ultrastructural features of adult liver cells (Poliak and
Shorey, 1967). Electron micrographs of 5-day embryonic liver cells
show that there is practically no endoplasmic reticulum present in the
cytoplasm. The amount of endoplasmic reticulum increases as develop
ment progresses, with smooth endoplasmic reticulum appearing prior to
rough endoplasmic reticulum.
There is a considerable amount of information about variations in
enzyme patterns during chick liver development. In 1967, Romanoff
(p. 106) listed 23 enzymes that have been studied in the embryonic
liver. Recently, Greengard and Thorndike (1974) reviewed studies on
52 enzymes in the same organ. They classified enzymes according to
the time of their emergence. The majority of enzymes emerge during
embryonic development. Some enzymes attain their mature level at the
arliest time test (about 10th day of incubation), while others are
.ill absent at the time of hatching. It is now well established that
each enzyme increases at its own rate, according to its own pattern.


10
The appearance of new enzymes results in new metabolic potentialities.
However, it is sometimes difficult to interpret the physiological role
of quantitative changes in enzyme levels.
Protein or albumin synthesis in chick embryos has been previously
studied in liver slices or in vivo. Duck-Chong et al. (1964) studied
the protein-synthesizing activity of chicken liver by incubating liver
slices of.8-, 14- and 20-day embryos and adult chicken with [^C]
leucine. They found that embryonic liver slices incorporated [^C]
leucine 15 to 30 times more rapidly than adult liver slices. In the
three different age embryos they studied, the rate of incorporation was
most rapid at 8-day and decreased as development proceeded. Reade et
al. (1965) injected ["* 'C] leucine into 14- to 20-day embryos and then
isolated various proteins from embryonic serum by electrophoresis. They
showed that label was incorporated into the albumin fraction. By
immunochemical techniques, Zaccheo and Grossi (1967) observed that
serum albumin could be detected in the circulating blood of the embryo
at the 4th day of development; however, no albumin was found in the
liver microsomal fraction until the 8th day of incubation. The yolk
sac is the only other embryonic tissue capable of producing albumin
(Butler, 1972); albumin thus appears to be supplied to the embryo from
the yolk sac before it is synthesized in the liver.
One approach to understanding the changes involved in the control
of protein synthesis has been the study of a cel I-free system. As
previously mentioned, the microscrne fraction isolated from chicken
liver was relatively inactive in amino acid incorporation (Campbell
and Kernot, 1959). Siler and Fried (1968) studied the poly (U)-


n
dependent polyphenylalanine synthesis in both homologous and heter
ologous systems composed of mixtures of ribosomes and cell sap
prepared from chicken and rat livers and found that with a given
ribosome preparation, chicken liver cell sap was much less active in
phenylalanine incorporation than rat liver cell sap. This relative
inactivity was ascribed to the presence of an uninhibited nuclease in
chicken liver cell sap. Zimmerman and Fried (1971) found that the
preparation of active ribosomes from adult and embryonic chicken
livers required a higher salt concentration (250 mM KC1) during
isolation than was common in the isolation of ribosomes from rat
liver (50 mM KOI). Jernigan et at. (1972) further studied the effect
of KOI concentration on the yield and the polyphenylalanine-synthe
sizing activity of chicken liver ribosomes. KC1 concentrations of
150-250 mM in the isolation medium resulted in higher ribosome yield
and activity than 25-50 mM. Polysomal amino acid-incorporating
systems have been prepared from chicken liver by Jernigan et at.
(1973). The high nuclease activity in chicken liver was minimized by
the use of bentonite and low molecular weight yeast RNA as nuclease
inhibitors. In the presence of such nuclease inhibitors, and under
appropriate ionic conditions, high molecular weight polysomes which
are active in protein synthesis were obtained. Thus the study of the
regulation of serum protein synthesis by components isolated from the
developing chicken liver is feasible.
Research Objectives
The purpose of this research is to study specific protein
synthesis during chick embryonic liver development with serum albumin


12
used as a probe for such study. As a first step in achieving this aim
the following questions were asked. (1) Does the protein synthesizing
activity of the components of a cell-free system derived from the livers
of chick embryos change during development? (2) Is there a correlation
between the structural differentiation of liver cells with the onset of
serum protein synthesis? (3) Can albumin synthesis be measured in the
cell-free system derived from embryonic chick livers? (4) Does albumin
synthesis measured in the cell-free system correlate with albumin
synthesis in vivo?


MATERIALS AND METHODS
Materials
Animals
Fertile white Leghorn eggs were obtained from the Poultry
Science Department of the University of Florida, and incubated, blunt
end up, at 38C in a Leahy model 624 electric incubator. The eggs
were turned three times a day. The age of the embryos was measured
from the start of incubation. Under these conditions of incubation,
the hatching time was 21 days.
Chemi cal s
3 3
Ribonuclease-free sucrose, [ H] lysine, [ H] leucine were purchased
from Schwartz-Mann. ATP, disodium salt; GTP, sodium salt; phosphoenol-
pyruvate, monopotassium salt; pyruvate kinase, Type II from rabbit
skeletal muscle; bovine pancreatic ribonuclease (Type XII-A); Torula
yeast RNA (Type VI); 2,5-diphenyloxazole and 1,4-bis-2(4-methyl-5-
phenyloxazole) were purchased from Sigma Chemical Company. P labeled
sodium pyrophosphate and Aquasol scintillation fluid were purchased from
New England Nuclear. Acrylamide and bisacrylamide were purchased from
Eastman Kodak Company. The more common organic or inorganic reagents
were analytical or reagent grade.
13


14
Biological Preparations
Preparation of Total Polysomes
Chick livers from embryos of the desired age were dissected out
G
and immersed in ice cold Buffer I (150 mM KC1, 8 mM MgC'l^ 20 mM Tris-HCl,
pH 7.4) containing 250 mM sucrose. The pooled livers were blotted dry
with paper towel, weighed and then homogenized in 3 volumes of ice-cold
Buffer I containing 250 mM sucrose, 5 mg/ml bentonite and 78.5 A^g units
(the absorbance at 260 nm)/ml yeast RNA at 120 rev./min by 4 strokes of
a glass-Teflon homogenizer. The homogenate was centrifuged for 15 min
at 12,000 X gmax in a Beckman-Spinco Model L centrifuge to ensure
complete removal of mitochondria (Figure 1). Nineteen parts of this
postmitochondrial supernatant were mixed with 1 part of 20% Triton
X-100, pH 7.4 and then 3 ml of this suspension were layered over a
discontinuous gradient of 3 ml and 2 ml of Buffer I containing 0.5 M
and 2.0 M sucrose respectively. The tubes containing these gradients
were centrifuged for 2.5 hours at 226,000 X g 5 in a Spinco Model L
iTlclX
centrifuge. The pellets were rinsed several times with Buffer I and
then resuspended in 0.25 ml Buffer I/ml original postmitochondrial
supernatant. The suspensions were then clarified by centrifugation at
12,000 X g for 10 min and used immediately for amino acid incorporation
studies. The concentration of soluble polysomes in such preparations was
about 20 Apgg units/ml. The ^60^280 ratl" suc^ polysome preparations
was in the range 1.77-1.87.


15
Liver homogenate in Buffer I containing 250 mM sucrose,
5 mg/ml bentonite and 78.5 Aggg units/ml yeast RNA
12,000 X g 15 min
Ilia a
Nuclei
Cell debris
Mitochondria
Postmitochondrial Supernatant
1. Make 1% in Triton
X-100
1. Make 1% in Triton
X-100 and 0.5 M in K
2. Layer over a
discontinuous
gradient of
Buffer I
containing
0.5 M and
2.0 M sucrose
2. Layer over a
discontinuous gradient
of Buffer II containing
0.5 M and 2.0 M
sucrose
Resuspend in
Buffer I
226,000 X gmax, 2.5 hr
(discard)
(discard) Total
polysomes
Resuspend in (discard)
Buffer II
Layer over 1.0 M
sucrose in Buffer
II
226,000 X g
2.5 hr
max
Resuspend in
Buffer I
(discard)
12,000 X gmax, 10 min
(discard)
Salt-washed
polysomes
Figure 1. Preparation of total and salt-washed polysomes.


16
Preparation of Salt-Washed Polysomes
The postmitochondrial supernatant prepared as described above was
treated with 1/19 volume of 20% Triton X-100 and made 0.5 M in K+ by
the addition of 2.5 M KC1 (Figure 1). The mixture was then layered
over a discontinuous gradient of 3 ml and 2 ml of Buffer II (500 mM KC1,
8 mM MgCl^, 20 mM Tris-HCl, pH 7.4) containing 0.5 M and 2.0 M sucrose
respectively. After centrifugation at 226,000 X g for 2.5 hours, the
ma x
pellet was rinsed and resuspended in Buffer II. The suspension was then
layered over 2 rnl of 1.0 M sucrose in Buffer II, and centrifuged again
at 226,000 X g^^ for 2.5 hours. The pellet was resuspended in Buffer I
and clarified by centrifugation at 12,000 X g for 10 min. This
suspension is designated "salt-washed polysomes."
Preparation of Free and Membrane-Bound Polysomes from Postnuclear
Supernatant
The preparation of free and total cytoplasmic polysomes from post-
nuclear supernatant followed the procedure described by Blobel and Potter
(1967) with some modification (Figure 2). The livers were homogenized in
3 volumes of Buffer I containing 250 mM sucrose, by 15 strokes at 120 rev./
min in a glass-Teflon homogenizer. One milliliter of the homogenate was
mixed with 2 ml of Buffer I containing 2.2 M sucrose. This was then
layered over 1 ml of 2.2 M sucrose in Buffer I. The sample was centri
fuged in a Spinco SW 50.1 swinging bucket rotor at 170,000 X g for
40 min. The supernatant was poured off and mixed with the material
adhering to the wall of the tube. This was rehomogenized manually by
4 strokes of a glass-Teflon homogenizer. The resulting solution was


17
Liver homogenate in Buffer I containing 250 mM sucrose
Make 1.55 M in sucrose
Layer over 1 ml of 2.2 M sucrose in Buffer I
170,000 X gmax> 40 min
Nuclei Cytoplasm
polysomes cytoplasmic
polysomes
Figure 2.
Preparation of total cytoplasmic and free polysomes
from postnuclear supernatant.


18
designated "postnuclear supernatant." For the preparation of free
ribosomes, 1 ml of postnuclear supernatant was layered over 3 ml of
2.0 M sucrose in Buffer I and the tube was filled with Buffer I con
taining 250 mM sucrose. The sample was centrifuged in a Spinco Ti
50 rotor at 150,000 X gm3v for 16 hours. The pellet contained the free
polysomes. For the preparation of total cytoplasmic polysomes, 1 ml
of the postnuclear supernatant was mixed with 0.4 ml of 20% Triton
X-100. Buffer I was added to it to provide a total volume of 8 ml.
The sample was centrifuged in a Spinco Ti 50 rotor at 150,000 X q
smax
for 4 hours. The pellet contained the total cytoplasmic polysomes.
Membrane-bound polysomes were determined by measuring the difference
between total cytoplasmic and free polysomes.
Preparation of Free and Membrane-Bound Polysomes from Postmi toehondrial
Supernatant
Polysomes were prepared from postmitochondrial supernatant by a
modification of the procedure (Figure 3) described by Blobel and Potter
(1967). The postmitochondrial supernatant prepared as described pre
viously was layered over a discontinuous gradient containing 2.0 ml and
3.0 ml of 2.0 M sucrose and 1.35 M sucrose respectively in Buffer I. The
gradient was centrifuged at 226,000 X gmax for 4 hours. Free polysomes
sediment in a pellet at the bottom of the tube whereas the membrane-
bound polysomes form a band at the boundary of the two sucrose layers.
The membrane-bound polysomes were removed from the top by suction. Free
polysomes were resuspended in Buffer I by homogenizing by hand. In some
experiments, the free and membrane-bound polysomes prepared under these
conditions were treated with 1/19 volume of 20% Triton X-100'and then


19
Liver homogenate in Buffer I containing 250 mM sucrose,
Nuclei
Cell debris
Mitochondria
Postmitochondrial supernatant
Layer over a discontinuous
gradient of Buffer I con
taining 1.35 M and 2.0 M
sucrose
226,000 X g 4 hr
smax
1.35 M sucrose
M/
Membrane-bound polysomes
2.0 M sucrose ?
Free polysomes
Figure 3. Preparation of free and membrane-bound polysomes from
postmitochondrial supernatant.


20
layered over 2.0 ml of 1.0 M sucrose in Buffer I. The gradients viere
centrifuged again in a Spinco Ti 50 rotor at 226,000 X g for 2.5
3max
hours.
Preparation of Cell Sap and Microsomes
One part of liver was homogenized in 3 to 6 parts of Buffer I
containing 250 mM sucrose at 120 rev./min by 10 strokes of a glass-Teflon
homogenizer (without bentonite and yeast RNA). The homogenate was
centrifuged at 12,000 X g(i,x for 15 min to prepare a postmitochondrial
supernatant. Cell sap was prepared by centrifuging the postmitochondrial
supernatant at 226,000 X g( for 1.5 hours. The resulting microsomal
pellet was resuspended in Buffer I containing 250 mM sucrose.
Preparation of 3,000 X g Supernatant
Livers were homogenized in 3 volumes of Buffer I containing 250 mM
sucrose at 120 rev./min by 10 strokes of a glass-Teflon homogenizer. The
homogenate was centrifuged at 3,000 X g for 10 min and the resulting
supernatant was removed from the centrifuge tube with a syringe.
Preparation of [^H] lysyl-tRNA
3
[ H] lysyl-tRNA was prepared from 1-week chick livers by the method
of von Ehrenstein (1967). Fifty grams of 1ivers were homogenized in 3
volumes of Buffer I containing 250 mM sucrose and then centrifuged at
12,000 X gmax for 15 min. The postmitochondrial supernatant was extracted
two times with an equal volume of buffer saturated phenol. Nucleic acids
were precipitated from the aqueous phase by adding 0.1 volume of cold


20% potassium acetate (pH 5) and 2.5 volume of -20C 95% ethanol and
held overnight at -20C. The precipitates were centrifuged and
extracted twice with 100 ml cold 1M NaCl. The supernatants from the
two extractions v/ere combined and tRNAs were precipitated by adding
2.5 volume of -20C 95% ethanol.
To discharge the amino acids, the tRNAs were incubated in 20 ml of
0.5 M Tris-HCl buffer (pH 8.8) for 1 hour at 35C and then reprecipi
tated by the addition of 0.1 volume of 20% potassium acetate (pH 5)
and 2.5 volume of -20C 95% ethanol.
The tRNAs thus obtained were further purified by dissolving in
10 ml of 0.1 M Tris-HCl buffer, pH 7.5, and applying the solution to a
DEAE cellulose column equilibrated with the same buffer. After washing
the column with 10 volumes of 0.1 M Tris-HCl buffer, pH 7.5, tRNAs were
eluted by 1 M NaCl solution in 0.1 M Tris-HCl, pH 7.5, and then pre
cipitated again by 2.5 volume of -20C 95% ethanol.
To charge the purified tRNAs with [ H] lysine, a final volume of
5 ml contained: 100 mM Tris-HCl (pH 7.5), 10 mM MgClo, 10 mM KC1, 2 mM
3
ATP, 1 mM dithioerythritol, 0.5 mCi ['H] lysine (specific activity 10
Ci/mmole), 0.2 mM of 19 other amino acids, cell sap (approximately 3.0
mg of protein from which endogeneous amino acids were removed by passing
through a Sephadex G-25 column), and 10 mg of purified tRNAs. Following
incubation at 35C for 20 min, the mixture was chilled in ice and extracted
with an equal volume of buffer saturated phenol. Charged tRNAs were pre
cipitated from the aqueous phase as described before. It was then
dissolved in H2O and dialyzed exhaustively against several changes of
H2O. The charged tRNAs were lyopnilized and stored at -20C.


22
Preparation of Nuclease Inhibitors
Bentonite was prepared by a modification of the procedure described
by Fraenkel-Conrat et al. (1S61). Fifty grams of bentonite were suspended
in 1 liter of a solution containing 8 mM MgC^ and 2 mM EDTA and then
stirred for 3 days. The bentonite was then washed by centrifuging
4 times in 10 mM Tris-HCl, pH 7.4, containing 8 mM MgCl^ each time
discarding the portion which sedimented in 5 min at 500 rev./min and
the portion which remained in suspension after 10 min at 8,000 rev./min.
The final 500 rev./min supernatant suspension (50 mg/ml) was frozen in
small portions and homogenized before use.
Yeast RNA (Sigma Type VI) was dissolved in H£0 by adding sufficient
Tris to maintain the pH at 7.0. The solution was centrifuged at 10,000
rev./min for 10 min, dialyzed at 4C once against 1% EDTA (pH 7.5) and
dialyzed exhaustively against 10 mM Tris-HCl. It was then centrifuged
for 4 hours at 226,000 X g and the supernatant was frozen in small
portions. The resulting solution had an A^q nm of 785 units/ml and the
RNA was of sufficiently low molecular weight that no detectable amount
was excluded by Sephadex G-100.
Biochemical Determinations
Amino Acid Incorporation
Incorporation of amino acids into hot trichloroacetic insoluble
material was assayed in a 0.5 ml volume at 35C. Except when rioted in
the text, each incubation tube contained: 4.48 A^q units/ml of poly
somes; 0.1 ml of cell sap (diluted as required with Buffer I); 0.05 ml of
*


23
3 ^
a mixture containing 12.5 yM [ H] lysine or [ H] leucine (125 yCi/ml)
and 10 yM of each of the other 19 amino acids, 20 mM Tris-liCl (pH 7.4),
150 mM KC1, 0.8 mM ATP, 0.2 mM GTP, 4 mM phosphoenolpyruvate, 1 mM
dithioerythritol and 20 yg/ml pyruvate kinase (approximately 10 yM
units). All solutions were kept on ice and were added to the incubation
tubes in a shaking water bath just prior to timing. Duplicate or
triplicate 0.1 ml samples were taken from the reaction mixture at
selected time intervals, spotted on 2.3 cm discs of Whatman 3MM.paper,
placed in cold 10% (w/v) trichloroacetic acid containing 0.2% unlabeled
lysine (or leucine) for at least 10 min. The paper discs were washed
according to the method originated by Mans and Novelli (1961) and
modified by Bollum (1966), including a 5 min wash in hot 5% trichloroacetic
acid, and then counted in a liquid scintillation counter in 5 ml of
toluene scintillation fluid (4 g/1 2,5-diphenyloxazole and 0.1 g/1
1,4-bis-2(4-methyl-5-phenyloxazolyl)-benzene) (Bray, 1960). The free
amino acids of the cell sap were measured on a Beckman 120C amino acid
analyzer. The specific radioactivity of each incubation mixture was
corrected according to the free amino acid content of each cell sap
sample.
[ H] lysyl-tRNA Incorporation
Cell sap was passed through a DEAE-Sephadex A-25 column prior
to assay. Incubations were in 0.5 ml and contained: 4.48 units/ml
of salt-washed polysomes, 0.1 ml cell sap, 20 mM Tris-HCl (pH 7.4), 6 mM
MgCl2* 1 dithioerythritol, 150 mM KC1, 0.2 mM GTP and 42,000 dpm
3
[H] lysyl-tRNA (100 yg tRNA). The reaction mixture was incubated at


24
35C. At selected intervals, duplicate 0.1 ml samples were spotted
on Whatman 3MM filter paper discs which were then placed in ice-cold
10% trichloroacetic acid containing 0.2% unlabeled lysine, washed and
counted as described before.
Aminoacyi-tRNA Synthetase Assay
Ami noacyl-tRNA synthetase activity was assayed by the amino acid-
32 32
dependent exchange of P between P labeled pyrophosphate (PPi) and
ATP according to the method of Stulberg and Novelli (1962), with some
modification. The incubation mixture contained the following components
in final volume of 1 ml: 0.1 ml cell sap, 100 mM Tris-HCl (pH 7.4),
5 mM MgCl^> 2 mM ATP, 2 mM 32PPi (104-l05 dpm), 50 mM KF, 1 mM dithio-
erythritol and 2 mM of each of 20 amino acids. The reaction was
initiated by the addition of cell sap and incubated at 35C for 15
min. The reaction was terminated by the addition of 0.5 ml of 7%
HCIO^ followed by 0.2 ml of a suspension of Norit A (200 mg dry weight).
Four milliliters of 0.1 M sodium acetate were added to the mixture which
was then mixed thoroughly with a wooden applicator stick. The adenosine
phosphates were absorbed by the Norit A and inorganic phosphates were
left in the solution. Norit A was separated by low-speed centrifugation,
resuspended in 5 ml of 0.5 M acetate buffer plus 0.1 M PPi (pH 4.5),
mixed thoroughly, centrifuged and the supernatant discarded. The wash
ing procedure was repeated 3 times with 5 ml of 0.05 M acetate, pH 4.5
(no PPi) and once with 5 ml of H^O. The Norit was then suspended in
5.0 ml of IN HC1 and heated at 100C for 15 min to hydrolyze the terminal
phosphate groups of ATP. After hydrolysis, the mixture was centrifuged


25
and 1 ml aliquots of the supernatant were removed and then counted in
a liquid scintillation counter.
Ribonuclease Assay
The ribonuclease assay is essentially a modification of the
procedure of Anfinsen et al. (1954), a method based on the liberation
of acid-soluble oligonucleotides from yeast RNA. The RNA substrate
was purified by the method of Utsunomiya and Roth (1955). Yeast RNA
(Sigma Type VI) was dissolved in H^O and the pH was adjusted to 7.0 by
adding Tris. The solution was dialyzed against 5 changes of 2.5
volumes of 0.025 M EDTA (pH 7.0) and then against 5 changes of 5.0
volumes of H^O. The concentration of the dialyzed RNA was adjusted
to 1% as determined by absorbance measurement. The purified RNA thus
obtained was stored frozen until use and this material gave very low
blank value. Ribonuclease activity was measured by incubating, at 35C
for 1 hour, 0.4 ml of 1% purified yeast RNA, 0.1 ml of 200 mM Tris-HCl
(pH 7.4) and 0.5 ml of enzyme preparation. Triplicate samples were
assayed when possible. The reaction was terminated by the addition of
0.5 ml ice-cold 0.75% uranyl acetate in 25% HC10^. The mixture was
kept at QC for at least 15 min. After centrifugation at low speed,
the supernatant was diluted 10 times with H^O and the absorbance at
260 nrn was measured in a Beckman DU spectrophotometer against a zero
time blank (containing both enzyme sample and RNA). Another blank
without enzyme was treated in the same way in order to correct for non-
enzymatic depolymerization of RNA.


26
Ribonuclease Inhibitor Assay
Ribonuclease inhibitor activity was measured according to the
method of Roth (1956) with some modification. A final volume of 1.0
ml contained: 0.1 ml of a solution containing 0.0125 yg of pancreatic
ribonuclease, 0.4 ml of 1% purified yeast RNA, 0.1 ml of 200 mM
Tris-HCl (pH 7.4) and 0.4 ml of cell sap. The mixture was incubated
at 35C for 60 min and the reaction was terminated by the addition of
0.5 ml of 0.75% uranyl acetate in 25% HC1C%. The A^-g of the sample
was measured as described previously. Controls containing 0.4 ml of
Buffer I instead of cell sap, and others containing 0.1 ml of H£0 in
place of pancreatic ribonuclease were assayed simultaneously.
Analysis of Cell-Free Reaction Products
For analysis of protein products, a 2-5 ml reaction mixture was
used. Each ml of reaction mixture contained 5-10 ^60 un^s f membrane-
bound or free polysomes, 1 mg cell sap protein, 20 mM Tris-HCl (pH 7.4),
100 mM KC1, 6 mM MgC^ 0.8 mM ATP, 0.2 mM GTP, 4 mM phosphoenolpyruvate,
20 yg pyruvate kinase, 1 mM dithioerythritol, 1 yM each of [ H] lysine
and [~H] leucine (12.5 yCi per ml reaction mixture), 1 yM of each of
the other 18 amino acids. Incubation was performed at 35C for 60
min. Subsequent treatment of the sample was carried out essentially
by the method of Koga and Tamaoki (1974) At the end of the incubation,
the reaction mixture was centrifuged at 150,000 X g for 1.5 hours
max
to sediment polysomes. The supernatant was dialyzed extensively against
several changes of 20 mM Tris-HCl (pH 7.4) and then subjected to poly-


27
acrylamicte gel electrophoresis. The pellet was resuspended in the
original volume of 20 mM Tris-HCl (pH 7.4). The sample was frozen in
dry ice-acetone, thawed and sonicated for 5 min. This treatment was
repeated three times. The disrupted sample was centrifuged at 150,000
X g for 1.5 hours and the resulting supernatant was dialyzed as
above and subjected to gel electrophoresis. Duplicate 10 yl samples
were taken from the reaction mixture in each step and spotted on filter
paper discs. Hot trichloroacetic acid insoluble materials were determined
as previously described.
In case of the 3,000 X g supernatant system, incubation for
amino acid incorporation was the same as with the polysomal system except
that 0.2 g equivalent of liver per ml reaction mixture was substituted
for polysomes and cell sap. Subsequent analysis of the reaction products
was as described above.
Polyacrylamide Gel Electrophoresis
Gel electrophoresis in the absence of SDS was performed in 7.5%
gels (acrylamide:bisacrylamide, 37.5:1) at pH 8.9 according to the method
of Davis (1964). Between 5,000 to 15,000 cpm of hot trichloroacetic acid
insoluble material was applied to the gels. The current was adjusted to
2 m Amp per tube for the first 20 min, then it was increased to 5 m Amp
per tube. Total running time was 2 hours. After electrophoresis, the
gels were sliced into 2 mm sections which were dissolved by incubating
in 0.2 ml of 30% H2O2 at 60C in tightly capped scintillation vials and
then counted in 10 ml of Aquasol scintillation fluid.
SDS-polyacrylamide gel electrophoresis was performed in 7.5% gel
at pH 7.1 according to the method of Palmiter et al. (1971). The current


28
was adjusted to 2 m Amp per tube for the first 20 min, then it was
increased to 5 m Amp per tube. Total running time was 4 hours. Radio
activity in the gel was determined as described above.
Immunoprecipitation
A monospecific antiserum to chicken serum albumin was produced in
a goat by three alternate weekly intramuscular injections of purified
chicken albumin. Fifty microliters of the antiserum would precipitate
between 6 to 12 yg of albumin at the equivalence point. For immuno
precipitation of the cell-free reaction products, 10 yg of purified
albumin were mixed with 100-200 yl of sample followed by the addition
of 100 yl of antialbumin serum. The mixture was incubated at 35C for
60 min and then at 4C for 24 hours. The sample was centrifuged at
low speed for 10 min at 4C. The precipitate was washed three times
with 20 mM Tris-HCl (pH 7.4) in 150 mM NaCl, and dissolved in 1% SDS,
]% g-mercaptoethanol and 20 mM Tris-HCl (pH 7.4) by boiling for 5 min.
Sucrose to a final concentration of 10% and Bromphenol blue tracking
dye were added to the sample and SDS gel electrophoresis was performed
as described above.
For assay of albumin synthesis, immunoprecipitation was performed
as described above except in the presence of 1% sodium deoxycholate and
1% Triton X-100 to reduce nonspecific precipitation (Shafritz, 1974b). The
immunoprecipitate was washed three times with 20 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 % sodium deoxycholate and 1% Triton X-100 and then dissolved
in 0.04 N NaOH. The radioactivity of this solution was determined by
counting in 10 ml of Aguasol scintillation fluid. A blank containing


29
nonimmune goat serum was treated under the same conditions and its
radioactivity subtracted from the radioactivity in the immuno-
precipitate.
Sucrose Gradient Centrifugation
Linear sucrose gradients (36 ml, 15-40% sucrose in Buffer I)
were prepared in an ISCO, Model 570, gradient former. Approximately
2.0 to 5.0 ApgQ units of polysomes were applied to each gradient and
centrifuged at 131,000 X g for 2 hours in a Spinco SW 27 swinging
bucket rotor. The bottom of each tube was punctured and the gradient
was displaced upwards with a 50% sucrose solution. The gradients were
monitored at 254 nm with an ISCO Model UA-4 ultraviolet analyzer.
Measurement of RNA and Protein
RNA concentration was determined by the method of Fleck and Munro
(1962). Protein was assayed by the method of Lowry et al. (1951) using
bovine serum albumin as a standard.
Statistical -Analyses
The results were expressed as mean value + the standard error when
more than three separate experiments were done. Other results were
expressed as the average of duplicate or triplicate determinations
when the values were derived from a single representative experiment.


RESULTS AND DISCUSSION
Characteristics of the Cell-Free Protein-Synthesizing System
Derived from Embryo Livers at Various Stages
of Development
Requirements of Cell-Free Amino Acid Incorporation
The cell-free amino acid incorporating system used in this
investigation was composed of polysomes and cell sap prepared from
chick embryo livers. Various conditions were tested to achieve
maximal activity, and the method of preparation outlined in Materials
and Methods was found to be most satisfactory. KC1 concentrations of
150-250 mM in the isolation medium resulted in higher polysome yield
and activity than 25 mM. In this study, 150 mM KC1 rather than 250
mM KC1 was used because higher KC1 concentration might increase the
risk of ribosome dissociation. Preliminary experiments showed poly
somes isolated in 150 mM KC1 to be slightly more active than those
isolated in 250 mM KC1. MgCl^ concentrations below 5 mM in the
isolation buffer reduced the yield of polysomes. The use of more
concentrated sucrose (2.0 M versus 1.5 M) in the lower layer of the
discontinuous gradients eliminated most of the protein impurities.
Polysomes isolated from livers of embryos older than 11 days showed
degradation by nuclease. Addition of bentonite and low molecular
weight yeast RNA to the isolation buffer as ruclease inhibitors
protected polysomes from degradation. Bentonite or yeast RNA alone
30


31
reduced the nuclease activity; however, a combination of the two
proved best.
Incorporation of amino acid into hot trichloacetic acid insoluble
material depended on the presence of polysomes, cell sap and an energy
source (Table 1). Figure 4 shows the effect of increasing amounts of
cell sap on amino acid incorporation by a fixed amount of polysomes.
Figure 5 represents another type of experiment in which the amount of
cell sap was fixed and increasing amounts of polysomes were added.
Within the concentration ranges tested, incorporation was linearly
dependent upon the amount of both cell sap and polysomes. The system
could not be saturated with cell sap probably because one or more
active components were in low concentration. Use of lower polysome
levels would have reduced the lysine incorporation to less than
significant levels. A typical time course of incorporation is shown
in Figure 6. The incorporation was linear with time for about 10
minutes.
Polysome Activity
The amino acid incorporating activity of cell-free systems derived
from embryos of different ages was compared. Polysomes prepared from
12-day and 19-day embryonic livers were incubated with homologous
cell sap. As shown in Figure 7, the cell-free system derived from
12-day embryos was found to be more active than that from 19-day
embryos. To investigate this difference further, the' activity of
polysomes derived from embryos of various ages was compared. For
this purpose, the assays were set up so that the polysome age was the


32
Table 1
Dependence of Protein Synthesis on Various
Components of the Reaction Mixture
Polysomes and cell sap were prepared from 17-day embryos and
incubated for 15 min at 35C in the complete reaction mixture
described, in Materials and Methods with 0.1 mg cell sap protein.
Incorporation of [3h] lysine was determined as acid soluble material
assuming 380 cpm = 1 pmol lysine incorporated. The values given are
the means of triplicate reaction mixtures assayed in triplicate.
System
pinoles of [^H] Lys
incorpd/mg rRNA
Complete
364.0
Complete
less polysomes
10.6
Complete
less cell sap
2.8
Comp!ete
less energy source*
3.4
ATP, GTP, Phosphoenolpyruvate


pnioles of l H] lys Incorpd
33
O
Figure 4. Effect of cell saD concentration on the [ H] lysine incor
poration into protein. Polysomes and cell sap were prepared
from 19-day embryos. Incubation was performed at 35C for
15 min as described in Materials and Methods with 0.2 mg
ribosomal RNA and indicated level of cell sap protein per
ml reaction mixture. Each point represents the average of
duplicate reaction mixtures assayed in triplicate.


pmoles of [ H] lys Incorpd
300
Polysomes and cell sap were prepared from 19-day embryos. Incubation was carried
out at 35C for 15 min as described in Materials and Methods with 1.0 mg cell sap
protein and indicated level of ribosomal RNA per ml reaction mixture. Each point
represents the average of duplicate reaction mixtures assayed in trip!icate.


35
10 15 20 25 30
Time (min)
Figure 6.
Time course of [ T;] lysine incorporation into protein.
Polysomes and cell sap rere prepared from 17-day embryos.
Incubation was performed as described in Materials arid
Methods except that 2.0 ml total volume was used. Each
ml reaction r¡ ture ine< l.Q g cell sap protein and
0.2 mg ribcsomal RNA. The values given ore the means of
duplicate reaction mixtures assayed in duplicate at each
time interval
time blan
nave
subtracted
from each point.


pinoles of [ H] lys Incorpd/mg rRNA
36
Figure 7. Amino acid incorporating activity of cell-free systems
derived from 12-day and 19-day embryos. Polysomes pre
pared from 12-day or 19-day embryonic livers were incubated
with homologous cell sap (1.0 mg cell sap protein per ml
reaction mixture) as described in Materials and Methods.
The incubation was for various times at 35C. The values
given are the means of triplicate reaction mixtures assayed
in duplicate, e, 12-day embryos; o, 19-day embryos.


37
only variable in the reaction mixture. Polysomes from embryos of
different ages viere prepared at the same time and incubated in the
presence of a standard cell sap preparation. As can be seen in
Figure 8, no significant differences in polysome activity were observed.
The sucrose gradient profiles of these polysome preparations are shown
in Figure 9. High molecular weight polysomes can be isolated from
embryonic livers throughout the developmental period studied. The
ratio of polysomes (10-30 ml from meniscus) to monomers (6 ml from
meniscus), indicated by the area under the profiles, decreased with
increasing developmental age. The polysome peak was found to be
nearer the meniscus in sucrose density gradients with increasing age,
indicating a decrease in average polysome length with maturity.
Since these polysomes were isolated, resuspended and then analyzed
by sucrose gradients, subtle differences in the profiles may be due
to minor variations in the complex isolation procedure.
Cell Sap Activity
The activity of cell saps derived from embryos of various ages
was also compared using a standard polysome preparation. The con
centration of free amino acid in each cell sap preparation varied
quite significantly which caused significant changes in the specific
radioactivity of each reaction mixture. Therefore the free amino acid
concentrations of each cell sap sample were determined in a Beckman
amino acid analyzer and used for corrections in the specific radio
activities when calculating activities of different cell sap samples.
When such corrections were made, the incorporation of amino acid was


pmoles [H] Lys Incorpd/mg rRNA
200
150 h
100
50
1 1 i . t I
10 12 14 16 18
Embryo age (days)
Figure 8. Amino acid incorporation by total polysomes derived from
embryos at various stages of development. Polysomes were
isolated from five different age embryos on the same day.
Each polysome preparation was incubated with cell sap
prepared from 19-day embryos (1.0 mg cell sap protein/ml
reaction mixture) and other co~oonents as described in
Materials and Methods. Incubation was for 15 min at 35C
The values given are the means 21 S.E. of three samples of
each age.


Figure 9. Sucrose gradient profiles of total polysomes derived
from various age embryos. Polysomes were prepared as
described in Materials and Methods. Between 2 to 4 A
units of polysomes were layered on each 15-40% sucrose0
gradient. The gradients were centrifuged and monitored
as described in Materials and Methods. (A) 10-day poly
somes, (B) 12-day polysomes, (C) 15-day polysomes, (D)
17-day polysomes, (E) 19-day polysomes.


254
40
ml from meniscus


41
proportional to cell sap concentration in the range usually assayed.
In all assays of cell sap activity, duplicate determinations at three
different cell sap concentrations were done to assure that comparisons
were made at the linear part of the incorporation versus cell sap
concentration curve.
3
At the beginning of this study, [ H] lysine was used for all
incorporation studies. During the analyses of cell sap samples, two
interesting observations were made. First, the free lysine concentration
in the cell sap appeared to be age-dependent. Lysine concentrations
of 12-13 day and 18-20 day cell sap were always considerably lower
than the other days (Figure 10). In order to investigate whether the
concentrations of other amino acids were also age-dependent, complete
amino acid analyses were performed for all cell sap samples. Some
representative results are shown in Figure 10. The concentrations of
leucine, histidine and methionine was constant throughout development,
while those of other amino acids showed variations with age. The
patterns of arginine and lysine were similar in that the variations
in concentration were parallel.
The second observation was that the lysine peak from embryonic
cell sap, as eluted from the amino acid analyzer, was not symmetrical.
As can be seen in Figure 11, it appeared that another component of
cell sap eluted a little earlier than lysine did and formed a shoulder
on the lysine peak. Attempts to identify this other component by
comparing with several possible known compounds were unsuccessful.
Changing the pH of the eluting buffer did not further separate the
two components. It is interesting that the phenomenon is not observed


Figure 10. Free amino acid content of the cell sap derived from
embryos at various stages of development. Cell sap
proteins were precipitated with 10S trichloroacetic
acid and the resulting supernatant was analyzed on a
Beckman 120C amino acid analyzer. The values represent
the average of at least three determinations. The
lysine values were the average of 5 to 7 determinations.
, lysine; r, valine; , arginine; a, histidine;
& leucine; o, methionine.


Embryo age (cays)
CO
16.0


p
(B) 13-day
Figure 11. Lysine profiles from amino acid analysis. (A) 11-day cell sap, (B) 13-day cell sap,
(C) 15-day cell sap, (D) 18-day cell sap, (E) adult cell sap.


45
in cell sap from adult chicken liver. At this point, all that can be
said is that the cell sap from embryonic chick liver apparently contains
a ninhydrin-positive component which is either absent or present in very
low concentration in the cell sap from adult chicken liver.
To ascertain that the measurement of lysine concentration did not
3
introduce artifacts in the assay of cell sap activity, ['H] leucine
was used in some experiments. Figure 12 shows the cell sap activities
*3 o
at different stages of development using either ["H] lysine or [-H]
leucine. Cell saps from young embryos were more active in amino acid
incorporation than those from older embryos. Similar results were
obtained with either labeled amino acid. This indicates that correction
of the specific radioactivity by amino acid analysis is reliable.
Figure 13 shows the effect of cell sap concentration on the incorpor
ating activity of cell sap prepared from two different age embryos.
The age-related activity difference was observed at all concentrations
tested.
It is known that supernatant factors tend to bind to crude poly
somes and that they can be removed by washing polysomes with 0.5 M KC1.
The factors bound to crude polysomes may introduce a significant back
ground incorporation when cell sap concentration is limiting and this
background incorporation may interfere with cell sap activity measure
ments. To test this possibility, cell sap activity was studied using
0.5 M KCl-washed polysomes. The results are shown in Figure 12. In
corporation was reduced considerably as compared with that measured
using crude polysomes, but similar age-related changes were obtained.
The above experiments suggest that cell sap from young embryos
is more active in amino acid incorporation than that from embryos at


pmole [ H] Lys or [JH] Leu incorpd/mg rRNA
46
J J J i 1 L
10 12 14 16 18 20
Embryo age (days)
Figure 12. Amino acid incorporating activity of cell saps derived from
embryos of various ages. Cell saps prepared from livers of
different age embryos were incubated with a standard crude or
salt-washed polysome preparation at 35C for 15 min as described
in Materials and Methods. The activity was determined using
three different cell sap concentrations (between 0.5 and 1.5
mg protein per ml reaction mixture). The results have been
normalized to incorporation per ~g cell sap protein. Each point
represents the mean + S.E. of three separate experiments. The
activity was measured with either [H] lysine or [^H] leucine.
[^H] lysine incorporation, crude polysomes; o, [^H] leucine
incorporation, crude polysomes; r,, pH] lysine incorporation,
salt-washed polysomes; o> [^H] leucine incorporation, salt-
washed polysomes.


pmole [ H] Lys incorpord/mg rRNA
47
Cell sap (mg protein/ml reaction mixture)
Figure 13. Cell sap dependence of [ H] lysine incorporation. Polysomes
prepared from 16-day embryonic livers were incubated with
cell sap prepared from 9-day or 16-day embryonic livers under
the conditions described in Materials and Methods. Each point
represents the average of duplicate reaction mixtures assayed
in triplicate, o, 9-day cell sap; c, 16-day cell sap.


48
later stages of development. The results of Duck-Chong et at. (1964)
also showed that liver slices from young embryos incorporated amino
acid more rapidly than those from old embryos. Whether the decline
in amino acid incorporation reflects the in vivo protein synthesizing
capacity is not clear. One possible correlation seems to be with the
decrease in growth rate of liver during development (Romanoff, 1967, p.
267).
3
[ H] lysyl-tRNA Incorporation
Cell sap contains enzymes and other factors which are required
for different steps in protein synthesis. In the following experiment,
the ability of cell sap to transfer labeled amino acid from aminoacyl-
3
tRNA into polypeptide was tested. [ H] lysyl-tRNA was prepared by
3
charging chick liver tRNA with [ H] lysine. To avoid differential
3
dilution of added [ H] lysyl-tRNA with endogenous lysyl-tRNA, the
cell sap was passed through a DEAE-Sephadex A-25 column to remove the
endogeneous tRNAs. The extent of incorporation was assayed using a
salt-washed polysome preparation. Under these conditions, the in
corporation of radioactivity was linear with time for at least 15
minutes and was dependent upon the amount of cell sap added (Figures
14 and 15).
The incorporation by cell sap derived from embryos of different
ages is shown in Figure 16. Cell sap from young embryos was more
active in the transfer of amino acid into polypeptide than cell sap
from older embryos. This is consistent with the previous observation
when labeled amino acids were used for incorporation, although the age-
related differences were not exactly parallel.


49
Time (min)
3 3
Figure 14. Time course of incorporation of [ H] lysine from [ H] lysyl-
tRNA into polypeptide. Salt-washed polysomes prepared from
20-day embryonic livers were incubated with the cell sap
prepared from 13- or 20-day embryonic livers under the con
ditions described in Materials and Methods. Each ml
reaction mixture contained 1.0 mg cell sap protein. The
values given are the means of triplicate determinations,
o, 13-day cell sap; o, 20-day cell sap.


X dpm/mg rRNA
50
Figure 15. Effect of cell sap concentration on the incorporation of
[3h] lysyl-tRNA into polypeptide. Polysomes and cell sap
were prepared from 16-day embryonic livers. The reaction
conditions have been described in '-'aterials and Methods.
Each point represents the average of duplicate reaction
mixtures assayed in triplicate.


X dpm/mg rRNA
51
60
50
40
30
? 20
o
10
10 12 14 16 18 20
Embryo age (days)
3
Figure 16. Lysine incorporation from [ H] lysyl-tRNA into polypeptide by
cell saps derived from embryos of various ages. Cell saps
prepared from embryos of different ages were incubated with
a standard salt-washed polysome preparation as described in
Materials and Methods. The incorporation was determined
using three different cell sap concentrations (between 0.5
and 1.5 mg protein per ml reaction mixture). The results
have been normalized to incorporation per mg cell sap
protein. Each point represents the mean + S.E. of three
separate experiments.


Ami noacyl-tRNA Synthetase Activity
The ability of cell sap to activate amino acids was studied.
32
Ami noacyl-tRNA synthetase activity was assayed by the exchange of P
labeled pyrophosphate with the pyrophosphoryl moiety of ATP in the
presence of amino acids. There is some evidence that synthetases
are associated with large particles (Hampel and Enger, 1973). The
distribution of synthetase activity in both the cell sap and micro
somal fraction was studied. As shown in Table 2, while some activity
was found in the microsomal fraction, more than 90% of the synthetase
activity was found in the cell sap. Preliminary experiments were
performed by measuring the synthetase activities of different age
embryos using both postmitochondrial supernatant and cell sap.
Parallel results were obtained. Therefore, in the following assay
cell sap was used as the source of enzyme.
While the pyrophosphate exchange reaction is dependent on the
presence of added amino acids (Table 3), there was a high background
exchange between pyrophosphate and ATP in the absence of added amino
acids. Dialysis of the sample reduced the background considerably.
After subtracting the blank without added amino acids, the activity
of the sample before and after dialysis was essentially the same.
The time course of pyrophosphate exchange is shown in Figure 17.
The reaction was linear with time up to 20 minutes. The exchange
reaction is linearly dependent upon the cell sap concentration thus
permitting the comparison of synthetase activity under these assay
conditions (Figure 18).


53
Table 2
Subcellular Distribution of Ami noacyl-tRNA
Synthetase Activity
The cell fractions were prepared from 19-day embryonic livers
by homogenizing livers in 5 volumes of Buffer I containing 250 mM
sucrose. Postmitochondrial supernatant and cell sap were prepared
as described in Materials and Methods. The microsomal pellet was
resuspended in the original volume of Buffer I, 0.1 ml of each
fraction was assayed as described in Materials and Methods.
Cell fraction
Synthetase Activity
nmole 32pp-¡ incorpd into ATP/15 min
Postmitochondrial supernatant
378
Cell sap
357
Microsomal pellet
32


54
Table 3
Amino Acid Requirement of the Pyrophosphate-ATP
Exchange Reaction
Cell sap prepared from 19-day embryos was divided into two
portions. One portion was dialyzed overnight at 4C against Buffer I
containing 250 mM sucrose. The other portion was stored at 4C
overnight. Pyrophosphate-ATP exchange was assayed with and without
added amino acid mixture as described in Materials and Methods.
32
nmole PPi incorpd into
ATP/15 min/mg protein
Cell sap 112
Cell sap + amino acid mixture 658
Cell sap (dialyzed) 44
Cell sap (dialyzed) + amino acid mixture 536


nmole PPi incorpd into ATP/mg cell sap protein
55
Figure 17. Time course of pyrophosphate-ATP exchange assay. Cell sap was
prepared from 17-day embryonic livers and was dialyzed overnight.
The reaction conditions have been described in Materials and
Methods. Approximately 500 pg cell sap protein/ml reaction
mixture was used to determine tre time course. Duplicate 1 ml
reaction mixtures were assayed at each time interval. Blanks
without added amino acid have been subtracted from each point.


56
Figure 18. Effect of cell sap concentration on the pyrophosphate exchange
reaction. Cell sap was prepared from 17-day embryonic livers
and was dialyzed overnight. The reaction conditions have been
described in Materials and Methods. Each point represents the
average of duplicate determinations, e, incubation with added
amino acid mixture; o, incubation without added amino acid
mixture.


57
Figure 19 shows the variation of ami noacyl-tRNA synthetase
activity of cell saps prepared from embryos between 9 and 20 days
of development. Synthetase activity seems to be somewhat higher in
the cell sap derived from embryos at later stages of development.
However, the activity difference throughout the development stages
we studied was less than 20%.
From the results, it becomes obvious that the ami noacyl-tRNA
synthetase activity in the cell sap can not account for all the decrease
in cell sap activity in supporting protein synthesis during development.
Based on the results of this experiment and the one on the incorporation
3
of [ H] ami noacyl-tRNA, it seems reasonable to assume that the decrease
in cell sap activity is probably due to a cell sap component involved in
a stage of protein synthesis different from and possibly subsequent to
the aminoacylation of tRNA.
Ribonuclease Activity
Ribonuclease activity in various subcellular fractions of embryonic
livers at different stages of development was assayed. The results are
summarized in Table 4. Ribonuclease activity in whole liver homogenate
almost doubled between 8 and 15 days of development. The particulate
fraction contained most of the cellular ribonuclease activity. The
changes in activity of the particulate fraction with development paralleled
those of the whole cell homogenate. Before the 12th day, no ribonuclease
activity could be detected in the cell sap. However, this activity in
creased 8-fold from day 12 to day 19. Some activity was present in
the microsomal fraction and it increased 5-fold from day 8 to day 19.


nmole PPi incorpord into ATP/mg cell sap protein.
58
20C
100
L _J 1 -Ju-
IO 12 14 16 18 20
Embryo age (days)
Figure 19. Ami noacyl-tRNA synthetase activity of cell sap derived from
embryos of various ages. The cell saps were dialyzed over
night and pyrophosphate-ATP exchange assayed was performed
as described in Materials and Methods. Incubation was for
15 min at 35C. The values represent the mean + S.E. of
3 to 5 separate experiments.


Table 4
Ribonuclease Activity in Liver Subcellular Fractions Derived
from Embryos of Various Ages
Livers were homogenized in 9 volumes of Buffer I containing 250 mM sucrose. The homogenate
was centrifuged at 12,000 X gmax for ^5 min. The pellet was resuspended in the same volume of
Buffer I containing 250 mM sucrose and designated "particulate fraction." Cell sap and micro
somal fractions were prepared from the supernatant. Ribonuclease activity was assayed as
described in Materials and Methods. The results are arbitrarily expressed as the difference in
A260 between samples and blanks. The values shown have been normalized to relative AA26O Per
gram liver.
Embryo age
(days)
Homogenate
Particulate
fraction
aA260
Microsome
Cell sap
8
2.30
2.19
0.031
0.015
12
3.11
3.27
0.078
0.011
15
4.15
4.01
0.110
0.067
19
4.08
4.55
0.159
0.128 '
(_n
LO


60
Measurement of cell sap inhibitor activity against bovine pan
creatic ribonuclease showed that no inhibition could be .detected in
the cell sap derived from embryos before 15 days of age (Table 5).
Twenty to thirty percent inhibition was observed after day 17. Similar
levels of inhibition were seen when the inhibitor activity was assayed
against the endogeneous ribonuclease in the liver particulate fraction.
The amount of inhibition observed in the older embryos was barely within
the range of significance. Many mechanisms could be responsible for this
small decrease in ribonuclease activity. One would be the presence of a
specific inhibitor. Another possible explanation would be that such
inhibition was nonspecifically caused by the adsorption of added ribo
nuclease to some cell sap component. The third reason may be that the
assay method is not sensitive enough to detect changes in the endo
nuclease activity. Since the amount of substrate RNA used in the assay
was in large excess, it is less likely that the inhibition was due to
competition for the substrate. The results are in line with that of
Roth (1962). He reported 30-60% inhibition for pancreatic ribonuclease
by adult chicken liver high speed supernatants under similar assay
conditions, but no inhibition by supernatant from 8-day and 9-day
whole embryos.
Increase in cellular protein synthesis has been demonstrated to
be associated with decreased ribonuclease activity and/or increased
ribonuclease inhibitor activity, and vice versa (Shortman, 1962;
Arora and de Lamirande, 1967; Moriyama et at. 1959; Sheppard et al.,
1970). It seems possible that the increase in ribonuclease activity
of cell sap with increasing developmental age may be connected with the
decrease in amino acid incorporating activity of the cell sap.


61
Table 5
Ribonuclease Inhibitor Activity in Cell Sap Derived
from Embryos of Various Ages
Livers were homogenized in 9 volumes of Buffer I containing 250
mM sucrose. The reaction mixture in 1 ml contained 0.0125 yg bovine
pancreatic ribonuclease or 0.1 ml particulate fraction (resuspended
in same original volume), 0.4 ml cell sap, 0.4 ml of 1% yeast RNA
and 0.1 ml of 200 mM Tris-HCl (pH 7.4). The mixture was incubated
at 35C for 60 min and precipitated with 0.5 ml of 0.75% uranyl
acetate in 25% HCIO4 and treated as described in Materials and Methods.
Each value represents mean + S.E.
Embryo age
(days)
% inhibition
Pancreatic RNase Particulate Rnase
8
12
15
17
19
20
2 + 4
4 + 3
10 + 4
21+6
27 + 5
24 + 8
6 + 4
25 + 8


62
Distribution and Activity of Free and
Membrane-Bound Polysomes
Distribution of Free and Membrane-Bound Polysomes
Since serum proteins are probably synthesized by membrane-bound
polysomes and early embryonic liver cells contain very little endo
plasmic reticulum, we looked for a correlation between the distribution
of membrane-bound and free polysomes and changes in protein synthetic
capacity during development. The amount of membrane-bound and free
polysomes was estimated by a modification of the method of Blobel and
Potter (1967). Because a significant amount of membrane-bound poly
somes tends to sediment in the mitochondrial fraction, total cytoplasmic
polysomes and free polysomes are separated from postnuclear supernatant.
Membrane-bound polysome content was determined as the difference between
total and free polysome content thus circumventing incomplete recovery
of membrane-bound polysomes from the boundary of two sucrose layers.
Usually the nuclear fraction contained 7-10% of the total cellular
RNA.
The total polysome content per gram of liver at various ages v/as
relatively constant (Figure 20). The free polysome content decreased
markedly between day 8 and day 18, whereas the membrane-bound polysome
content increased during the same period. Hence the percentage of total
polysomes present on membranes increased 2-fold as development progressed.
To ascertain that ribosomes did not readsorb the membrane during
isolation, a mixing experiment was performed. Postnuclear supernatant
was prepared from a 1:1 mixture of 10-day and 17-day liver homogenates.
Free and membrane-bound polysome contents were measured in this mixture


Figure 20. Distribution of free and membrane-bound polysomes in
livers at various stages of development. Livers were
homogenized in 3 volumes of Buffer I containing 250 mM
sucrose. Free and membrane-bound polysomes were
fractionated from the postnuclear supernatant as described
in Materials and Methods. The values are the means +
S.E. of 3 to 4 separate experiments. The results were
expressed in mg RNA per ml homogenate, o, total polysomes;
e, free polysomes; A, membrane-bound polysomes; A, %
membrane-bound in total polysomes.


mg RNA
64
Embryo age (days)


as well as in the 10-day and 17-day controls. The amount of free and
membrane-bound polysomes found in the mixture was approximately the same
as the average of the two controls (Table 6). Therefore, reassociation
of free ribosomes with the membrane during isolation was not significant
These experiments indicate that the proportion of membrane-bound
polysomes in total polysomes increases during development. Chemical
measurements by Poliak and Ward (1967) showed that the phospholipid
content of the microsomal fraction increases during development. An
increase in rough endoplasmic reticulum seems to be a general phenomenon
in cells undergoing rapid growth such as in the neonatal (Dallner et at.
1966) and regenerating rat liver (Tata, 1970). The exact function of
the ribosome-membrane assembly is not clear, since an accumulation of
intracellular protein also occurs during the same period. Because of
the increase in serum protein concentration in blood during development,
it seems highly probable that the increase in membrane-bound polysomes
in embryonic chick liver results in the increased synthesis of secreted
proteins.
/Activity of Free and Membrane-Bound Polysomes
The preceding experiments suggest that the distribution of free
and membrane-bound polysomes changes while the amino acid incorporating
activity of total polysomes remains the same during development. It
was not known whether the activity of the free and membrane-bound
polysomes is the same or whether their activities change during develop
ment. To resolve this question, the amino acid incorporating activity
of these two types of polysomes was assayed.


Table 6
Distribution of Free and Membrane-Bound Polysomes in Homogenates
of 10-day and 17-day Mixture
Livers were homogenized in 3 volumes of Buffer I containing 250 mM sucrose. Postnuclear
supernatant was prepared from 1:1 mixture of 10-day and 17-day liver homogenates. Free and
membrane-bound polysomes were fractionated as described in Materials and Methods. The values
represent the average of 3 determinations in a single experiment. The results were expressed
in mg RNA in 1 ml homogenate.
Embryo age
days
Total polysomes
mg RNA
Free polysomes
mg RNA
Membrane-Bound
polysomes
mg RNA
Bound/Total
polysomes
%
10
1.92
1.13
0.79
41
17
1.81
0.63
1.18
65
10 + 17
1.88
0.92
0.96
51


67
For studying ami no acid incorporation, membrane-bound and free
polysomes viere isolated from postmitochondrial rather than postnuclear
supernatant, because polysomes were more degraded in the postnuclear
supernatant. The disadvantage of the procedure used is that con
siderable losses of membrane-bound polysomes occur during separation
of postmitochondrial supernatant.
The amino acid incorporating activity of free and membrane-bound
polysomes prepared from 12-day, 15-day and 19-day embryonic livers is
shown in Table 7. Because of the small size and low membrane-bound
polysome content of liver, polysome analyses from embryos younger
than 12 days were not significant. The activity of free polysomes
derived from embryos of these three ages was approximately the same.
In contrast, the activity of membrane-bound polysomes appeared to
decrease with age. The activity of free polysomes was found to be
higher than that of membrane-bound polysomes derived from embryos of
the same age. The difference between the activities of the two
types of polysomes was more pronounced in the older embryos compared
with that in young embryos. However, polysomes could be released from
the 19-day membrane-bound polysome preparation by detergent treatment
(Figure 21). The greater amount of ultraviolet absorbing material in
the upper fractions of the membrane-bound versus free polysome prepa
rations is probably membranous material released upon detergent treat
ment. The time course of incorporation showed that these two types of
polysomes incorporated amino acid at the same initial rate, however;
with membrane-bound polysomes it leveled off earlier than that with


Table 7
Amino Acid Incorporating Activity of Free and Membrane-Bound Polysomes
Before and After Triton X-100 Treatment
Free and membrane-bound polysomes were prepared and assayed as described in Materials and
Methods. Incubation was for 30 min. The values represent mean + S.E. from 3 to 4 experiments.
Before Triton X-100
Treatment
After Triton X-100
Treatment
Free Bound
Free Bound
polysomes polysomes
Free/Bound
polysomes polysomes
Free/Bound
Embryo
age
pmole [ H] lysine
pmole [ H] lysine
days
incorpd/mg rRNA
%
incorpd/mg rRNA
%
12
423
+
36.1
348
+
31.8
82
360
+
24.9
327
+
35.4
91
15
396
+
28.8
285
+
23.7
72
354
+
36.7
345
+
28.5
97
19
384
+
52.2
204
+
48.9
53
318
+
39.6
336
+
45.1
106


Figure 21. Sucrose gradient profiles of membrane-bound and free
polysomes prepared from 19-day embryonic livers without
using detergent. Polysomes were analyzed after addition
of detergent to the membrane-bound polysome preparation
as described in Materials and Methods. Approximately 3.0
A26O units of polysomes were layered over 36 ml 15-40%
sucrose gradients centrifuged and monitored as described.
, represents free polysomes; represents
membrane-bound polysomes.


70
0.4
0.3
vf-
LO
CM
C
0.2
0.1
10
20
ml from meniscus
I L
30


71
free polysomes (Figure 22). Once again the difference was greater in
older embryos.
In the-above experiment, the two types of polysomes were prepared
without using detergent. Because the membrane was still associated with
polysomes during the assay of amino acid incorporation, the observed
difference in activity could not be attributed solely to the polysome.
In other words, the presence of membrane may be inhibitory to amino
acid incorporation. To test this possibility, both free and membrane-
bound polysomes were treated with Triton X-100 and reisolated. To
obtain undegraded polysomes, yeast RNA had to be added to the polysome
preparation during detergent treatment. In the absence of yeast RNA,
membrane-bound polysomes were degraded extensively as shown by the
sucrose gradient profiles in Figure 23. Unlike the finding with rat
liver (Blobel and Potter, 1966), addition of cell sap to the membrane-
bound polysomes during Triton X-100 treatment did not protect polysomes
from degradation (Figure 23B). In contrast, relatively undegraded
free polysomes were isolated in the absence as well as the presence
of yeast RNA (Figure 24). It appeared that the detergent treatment
might release or activate some ribonuclease present in the polysome
preparations. Therefore, ribonuclease activity in the polysome prep
arations was assayed (Table 8). Before Triton treatment, the ribo
nuclease activity in free polysomes from 18-day embryos was low (just
twice the detectable level in this assay). That in the membrane-bound
polysomes was 5-fold higher. After membrane was removed by detergent,
the polysomes originally bound to the membrane contained one third
their original ribonuclease activity. Since more ribonuclease activity


maximum incorporation of [ H] lysi
o 100
80
60
40
20
Figure 22.
Time courses of amino acid incorporation of membrane-bound and free polysomes. Membrane-bound and
free polysomes were prepared from 12-day and 19-day embryonic livers without using detergent.
Polysomes were incubated with homologous cell sap and assayed as described in Materials and Methods. ^
Incorporation values were expressed as % maximum of free polysomes at 60 min. Separate maximum
incorporation values were used for 12-day and 19-day preparations. o, free polysomes; membrane-
bound polysomes.


Figure 23. Sucrose gradient profiles of membrane-bound polysomes
after Triton X-100 treatment. Membrane-bound polysomes
prepared from 19-day embryonic livers (approximately 20
A26O units/ml) were divided into three portions. Each
portion was treated with 20% Triton X-100 to a final
concentration of 1% (A) in the presence of 20 A260 units/
ml low molecular weight yeast RNA (B) in the presence of
cell sap (approximately 3 mg protein/ml) (C) in the
absence of yeast RNA and cell sap. Each portion was
layered over 2 ml of 1.0 M sucrose in Buffer I and
centrifuged at 226,000 X gmax for 2.5 hrs to resediment
polysomes. The pellets were resuspended in Buffer I
and approximately 4.0 A260 units of each sample were
layered on a 36 ml 15-40% sucrose gradient, centrifuged
and monitored as described.


74
ml from meniscus


Figure 24. Sucrose gradient profiles of free polysomes after Triton
X-100 treatment. Free polysomes prepared from 19-day
embryonic livers (approximately 20 A26O units/ml) were
divided into 2 portions. Each portion was treated with
20% Triton X-100 to a final concentration of 1% (A) in the
presence of 20 A26O units/ml of low molecular yeast RNA
(B) in the absence of yeast RNA. Each sample was layered
over 2.0 ml of 1.0 M sucrose in Buffer I and centrifuged
at 226,000 X gmax for 2.5 hrs to resediment polysomes.
The pellets were resuspended in Buffer I and approximately
6.0 A26O units of each sample were layered on a 36 ml 15-40%
sucrose gradient and centrifuged and monitored as described.


10 20
X L
L
30
ml from meniscus


77
Table 8
Ribonuclease Activity in Free and Membrane-Bound Polysomes
Free and membrane-bound polysomes were prepared as described in
Materials and Methods. Membrane-bound polysomes were treated with
20% Triton X-100 to a final concentration of 1%. The mixture was
layered over 2.0 ml of 1.0 M sucrose in Buffer I and centrifuged at
226,000 X gmax for 2.5 hours to sediment polysomes. The super
natant was diluted 4 times and centrifuged at 226,000 X gmax for 1
hour. The pellet thus obtained was resuspended in Buffer I of the
same volume as the original membrane-bound polysomes preparation and
designated "membranous material." Ribonuclease activity was assayed
as described in Materials and Methods. Incubation was at 35C for
60 min.
12-day
embryo
18-day
embryo
AA26Q'/m9 rRNA
Before Triton X-100 treatment
Free polysomes
0.043
0.061
Membrane-bound polysomes
0.144
0.306
After Triton X-100 treatment
Membrane-bound polysome
0.072
0.093
Membranous material
0.401


78
was found in the membranous material than was present with the membrane-
bound polysomes, the detergent appears to enhance the nuclease activity.
Similar results were obtained for polysomes derived from 12-day embryos
but the nuclease activity was much lower in all fractions as compared
with 18-day embryos. The results suggest that the ribonuclease
activity found in polysome preparations is associated with the membrane
rather than with the polysome. The small degree of degradation of
free polysomes upon detergent treatment shown in Figure 24 may be
due to some membrane contamination in the free polysome preparations.
The ^260/;^280 ra^"''os f the Triton X-100 treated free and membrane-
bound polysomes were 1.75-1.88 which indicated that the polysomes were
almost completely devoid of membrane. The amino acid incorporating
activity of the detergent treated polysomes is shown in Table 7. No
difference in activity was observed between free and membrane-bound
polysomes derived from embryos of the ages tested. This result suggests
that the activity difference observed between free and membrane-bound
polysomes before detergent treatment is probably due to some component
present in the membrane. Ribonuclease activity found in the membrane may
be that one component. The more rapid decay of amino acid incorporating
activity of membrane-bound polysomes as compared with that of free
polysomes is consistent with a membrane-bound nuclease active during
incubation for amino acid incorporation. The higher nuclease activity
in the membrane-bound polysomes of older embryos could also explain the
relative activity difference between membrane-bound and free polysomes.
It is interesting to note that detergent treatment has been shown to
alter the behavior of membrane-bound polyscr.es by other workers


79
(Bloemendal et al., 1967; McDonald and Konner, 1971). However, the
difference may also be ascribed to a different average size of the
mRNA molecules in the two polysomes.
The relative amino acid incorporating activity of membrane-bound
and free polysomes seems to depend upon the method as well as the ionic
conditions used for isolation of the two polysome fractions, since in
rat liver cells, membrane-bound polysomes have been reported to be
more (Tata and Wi11iams-Ashman, 1967; Pain et al., 1974), equally
(Bloemendal etal.> 1967; Takagi and Ogata, 1968), or less (Ragnotti,
Lawford and Campbell, 1969; McDonald and Konner, 1971) active than free
polysomes, apparently the result of different isolation procedures.
Under the assay conditions described above, we detected no difference
between the activity of free and membrane-bound polysomes. However,
membrane seems to affect amino acid incorporation by polysomes in the
cell-free system.
Albumin Synthesis
As a first step in our study of the molecular events that
initiate and control albumin synthesis during development, it was
decided to measure albumin synthesis in the cell-free system. Free and
membrane-bound polysomes were incubated for amino acid incorporation.
After incubation, the reaction mixtures were centrifuged to sediment
polysomes. Approximately 30-40% of the hot trichloroacetic insoluble
counts was released into the supernatant from the free polysomes and
15-20% of counts was released from the membrane-bound polysomes. The
supernatants from each reaction mixture were dialyzed extensively and


80
then subjected to gel electrophoresis at pH 8.9 in a 7.5% polyacrylamide
gel. Under these conditions, authentic chicken serum albumin migrates
faster than most other serum proteins so that it appears as a discrete
band behind the bromphenol blue tracking dye. Figure 25 shows the gel
patterns of the dialyzed supernatants derived from 16-day embryos.
No significant radioactive peak appeared to migrate at the position
of standard albumin. Finding no albumin in the supernatant, the
polysomal pellets were resuspended in buffer, frozen, thawed, sonicated
and centrifuged again. About 25-30% of the hot trichloroacetic acid
insoluble radioactivity was released into the supernatant by such
treatment. The released material was dialyzed and analyzed by gel
electrophoresis. Figure 26 shows the gel pattern of the materials
released from the free and membrane-bound polysomes derived from 16-day
embryos. In the free polysome preparation, some radioactivity was
present in the region corresponding to albumin standard, but no
discrete band was observed. One major peak of radioactivity from
the membrane-bound polysome preparation comigrated with standard albumin.
To characterize this peak further, carrier chicken serum albumin was
added to another aliquot of the sample and the mixture was reacted
with goat antiserum against chicken serum albumin. After immuno-
precipitation, the supernatant was subjected to gel electrophoresis.
The antiserum removed the peak of radioactivity which had comigrated
with albumin (Figure 27). The immunoprecipitate was washed several times,
dissolved, denatured with SDS and subjected to SDS polyacrylamide gel
electrophoresis (Figure 28). A radioactive band was observed comigrating
with standard albumin run on a sister gel. The radioactivity in the band


81
Figure 25. Polyacrylamide gel electrophoresis of cell-free protein products
released from membrane-bound and free polysomes before sonication.
Membrane-bound and free polysomes prepared from 16-day embryonic
livers were incubated for protein synthesis as described in
Materials and Methods. After incubation, the reaction mixture
was centrifuged to sediment polysomes and the supernatant was
dialyzed extensively and analyzed by gel electrophoresis as
described in Materials and Methods. Approximately 9000 cpm and
5000 cpm of trichloroacetic acid insoluble material from free and
membrane-bound polysomes respectively were applied to the gels.
The protein staining band of standard albumin is indicated in the
graph, o, free polysomes, e, membrane-bound polysomes.


Figure 26. Polyacrylamide gel electrophoresis of cell-free reaction
products released from membrane-bound and free polysomes
by sonication. Conditions for amino acid incorporation
and analysis of the products have been described in
Materials and Methods. About 7,000 to 15,000 cpm of
trichloroacetic acid insoluble material was applied to
the gels. The protein staining band of standard albumin
is indicated in the graph. A) 16-day membrane-bound
polysomes (B) 16-day free polysomes (C) 12-day membrane-
bound polysomes (D) 19-day membrane-bound polysomes.


(A)
(B)
Fraction No.
Fraction No.


84
Fraction No.
Figure 27. Polyacrylamide gel electrophoresis of cell-free protein products
of 16-day membrane-bound polysomes remaining after precipitation
with antiserum against albumin. The cell-free protein products
were incubated with antiserum against albumin. After removal of
the immunoprecipitate by centrifugation, the supernatant was
subjected to gel electrophoresis as described in Materials and
Methods. The dotted line is the profile of the radioactive
products present prior to immunoprecipitation. The protein
staining band of standard serum albumin is indicated in the
graph.


Figure 28. SDS-polyacrylamide gel electrophoresis of the cell-free protein
products precipitated with antiserum against albumin. The cell-
free products from 16-day membrane-bound polysomes were incubated
with antiserum against albumin. The immunoprecipitate was washed
and dissolved in 20 mM Tris-HCl (pH 7.4), 1% SDS and ]% 6-
mercaptoethanol. The sample was subjected to SDS-polyacrylamide
gel electrophoresis as described in Materials and Methods. The
protein staining band of standard serum albumin is indicated in
the graph.


86
represents more than 70% of total radioactivity recovered from the
gel. There was some radioactivity present in a lower molecular weight
region which may represent either incomplete albumin chains or non
specific precipitation. These experiments indicate that the major peak
of radioactivity represents newly synthesized albumin. The gel patterns
of products synthesized by membrane-bound polysomes derived from 12-day
and 19-day embryos are shown in Figure 26. They are very similar to the
16-day pattern.
To determine the amount of albumin synthesized, each sample
was reacted with antiserum against albumin and the resulting immuno-
precipitate was counted. Table 9 summarizes the results. The per
centages of albumin in total polypeptides synthesized by membrane-
bound polysomes were approximately the same at different ages. The
amount of albumin synthesized by free polysomes was much less than
that synthesized by membrane-bound polysomes. The result suggests
that membrane-bound polysomes are the major site for albumin synthesis
in embryonic chick liver cells. It is interesting that this phenomenon
is seen even in the 12-day embryonic livers which contain relatively
fewer membrane-bound polysomes. The 8- to 10-fold difference in albumin
synthesis by membrane-bound versus free polysomes is in the same range
as the results of similar investigations in mammalian systems (Shafritz,
1974b; Koga and Tamaoki, 1974).
Although the amount of albumin synthesized per weight of membrane-
bound polysomes remained constant during development, the possibility
remains that the increase in albumin synthesis in vivo is due to the
increase in the content of membrane-bound polysomes during development.


Table 9
Albumin Synthesis by Membrane-Bound and Free Liver Polysomes Derived
from Embryos of Various Ages
Conditions for amino acid incorporation
Materials and Methods. Each value represents
and
the
immunoprecipitation have been described in
average of 3 determinations.
Embryo age
Polysome
A1bumin
1
fotal TCA insoluble
Albumin as % of total TCA
(days)
cpm
polypeptides
insoluble polypeptides
cpm
12
free
655
116890
0.56
bound
4862
107380
4.52
16
free
564
129340
0.44
bound
4148
98570
4.21
19
bound
3760
86830
4.33
co


88
A cell-free system composed of low speed supernatant of liver homogenate
seemed to be suitable for testing this possibility for the following
reasons. The low speed supernatant contains both membrane-bound and
free polysomes present in their in vivo ratio. This system has been
shown to synthesize polypeptides with an in vivo-like pattern (Hill,
Wilson and Hoagland, 1972). Another advantage of this system is that
it requires only small liver samples which allows the study of early
embryos.
In the following experiments, the cell-free reaction products
synthesized by 3,000 X g supernatant derived from 9-day, 13-day and
16-day embryos were compared. After incubation for protein synthesis,
the reaction mixtures were centrifuged to sediment microsomes. The
supernatants of 9-day, 13-day and 16-day reaction mixtures so prepared
contained 35%, 23% and 17% of the total hot trichloroacetic acid in
soluble counts respectively. This is probably due to the presence of
different amounts of membrane-bound polysomes in the samples, because
a lower percentage of radioactivity is released from membrane-bound
polysomes as shown in the previous experiment and in the results of
other workers (Andrews and Tata, 1971). The supernatants did not
contain albumin as judged by gel electrophoresis and immunoprecipitation
(not shown). The microsomal pellets were frozen, thawed and sonicated.
The sonic supernatants were dialyzed and analyzed by gel electrophoresis.
The gel patterns are shown in Figure 29. In each sample, a radioactive
peak comigrated with standard albumin. The amount of albumin synthesized
in each sample was again estimated by counting the immunoprecipate after
reaction with anitserum against albumin. The relative proportion of
albumin synthesized by 9-day embryos was much less than that synthesized


Figure 29. Polyacrylamide gel electrophoresis of cell-free protein
products synthesized by 3,000 X g supernatant from livers
at various stages of development. Conditions for amino
acid incorporation and analysis of products have been
described in Materials and Methods. About 7,000 to
9,000 cpm of trichloroacetic acid insoluble material
was applied to each gel. The protein staining band of
standard albumin is indicated in the graph. (A) 9-day
embryos (B) 13-day embryos (C) 16-day embryos.


90
(A)
6 i
Fraction No.
Fraction No.


91
by 13-day and 16-day embryos (Table 10). The increase in the amount
of albumin synthesis with developmental age agrees with the increase
in albumin concentration in the serum. This result confirms the
previously postulated correlation between an increase in the proportion
of membrane-bound polysomes and an increase in specific protein synthesis.


Table 10
Albumin Synthesis by 3000 X g Supernatant Derived from Embryos of Various Ages
Conditions for amino acid incorporation and immunoprecipitation have been described in
Materials and Methods. Each value represents the average of 3 determinations.
Experiment
Number
Embryo age
(days)
A1 bumin
cpm
Total TCA insoluble
polypeptides
cpm
Albumin as % of total TCA
insoluble polypeptides
1
9
794
72320
1.1
13
2174
90040
2.4
16
2894
82390
3.5
2
9
403
44760
0.9
16
1379
47690
2.9
r\3


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'DOOQHU 6LHNHYLW] 3 DQG 3DODGH ( f &HOO %LRO 'DYLV % f $QQ 1 < $FDG 6FL 'XFN&KRQJ & 3ROLDN DQG 1RUWK 5 f &HOO %LRO (QJOH 5 / DQG :RRGV 5 f LQ 7KH 3ODVPD 3URWHLQV 3XWQDP ) : HGf &KDSWHU )OHFN $ DQG 0XQUR + 1 f %LRFKLP %LRSK\V $FWD )UDHQNHO&RQUDW + 6LQJHU % DQG 7VXJLWD $ f 9LURORJ\ *UHHQJDUG DQG 7KRUQGLNH f (Q]\PH +DPSHO $ DQG (QJHU 0 f 0RO %LRO +DVHONRUQ 5 DQG 5RWKPDQ'HQHV / % f $QQ 5HY %LRFKHP +HQGULNVHQ DQG 6DPL VRQ 0 ( f $UFK %LRFKHP %LRSK\V +H\ZRRG 6 0 f 1DWXUH +H\ZRRG 6 0 DQG 7KRPSVRQ : & f %LRFKHP %LRSK\V 5HV &RQPXQ +LFNV 6 'U\VGDOH : DQG 0XQUR + 1 f 6FLHQFH +LOO + = :LOVRQ 6 + DQG +RDJODQG 0 % f %LRFKLP %LRSK\ $FWD $OO +RDJODQG 0 % f %LRFKLP %LRSK\V $FLD +RDJODQG 0 % .HOOHU ( % DQG =DPHFQLN 3 & f %LRO &KHP +RDJODQG 0 % =DPHFQLN 3 & 6KDURQ 1 /LSPDQQ ) 6WXOEHUJ 0 3 DQG %R\HU 3 f %LRFKLP %LRSK\V $FWD ,ODQ DQG OLDQ f 'HYHORS %LRO -HUQLJDQ + 0 ODFHQD 0 $ DQG )ULHG 0 f %LRFKLP %LRSK\V $FWD -HUQLJDQ + 0 &KX 0 DQG )ULHG 0 f %LRFKLP %LRSK\V $FWD

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.DHPSIHU 5 f 3URF 1DWO $FDG 6FL 86 .DHPSIHU ) DQG .DXIPDQ f 3URF 1DWO $FDG 6FL 86 .RJD DQG 7DPDRNL 7 f %LRFKHPLVWU\ .UDIW 1 DQG 6KRUWPDQ f %LRFKHP %LRSK\V $FWD /DUQERUJ 0 5 DQG =DPHFQLN 3 & f %LRFKLP %LRSK\V $FWD /DQH & 0DUEDL[ DQG *XUGLQ % f 0RO %LRO /RZU\ & + 5RVHEURXJK 1 )DUU $ / DQG 5DQGDOO 5 f %LRO &KHP 0DQV 5 DQG 1RYHOO L f $UFK %LRFKHP %LRSK\V 0DULDQL $ 6SDGRQL 0 $ DQG 7RPDVVL f 1DWXUH 0F'RQDOG 5 DQG .RQQHU $ f )(%6 /HWWHUV 0RQUR 5 ( f 0RO %LRO 0RUL\DPD 7 8PHGD 7 1DNVKLPD 6 2XUD + DQG 7VXNDGD f %LRFKHP 0XQUR + 1 1LDVPLWK DQG :LNUDPDQ\DNH 7 : f %LRFKHP 3DLQ 9 0 f %LRFKLP %LRSK\V $FWD 3DLQ 9 0 DQG &OHPHQV 0 f )(%6 /HWWHUV 3DLQ 9 0 /DQRL[ %HUJHURQ 0 DQG &OHPHQV 0 f %LRFKLP %LRSK\V $FWD 3DODGH ( DQG 6LHNHYLW] 3 f %LRSK\V %LRFKHP &\WRO 3DOPLWHU 5 2ND 7 DQG 6FKLPNH 5 7 f %LRO &KHP 3HWHUV 7 DQG $QILQVHQ & % f %LRO &KHP 3HWHUV /RJDQ $ & DQG 6DQIRUG & $ f %LRFKLP %LRSK\V $FWD

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3HWHUV 7 f +LVWRFKHP &\WRFKHP 3ROLDN DQG 6KRUH\ & f $YVW H[S %LRO PHG 6FL 3ROLDN DQG :DUG % f %LRFKHP 5DJQRWWL /DZIRUG 5 DQG &DPSEHOO 3 1 f %LRDKHP 5HDGH 3 & -HQNLQ & 5 DQG 7XUQHU f $XVW H[S %LRO PHG 6FL 5HGPDQ & 0 f %LRDKHP %LRSK\V 5HV &RPQLP 5KRDGV 5 ( 0F.QLJKW 6 DQG 6FKLQLNH 5 7 f %LRO &KHUQ 5RPDQRII $ / f %LRFKHPLVWU\ RI WKH $YLDQ (PEU\R ,QWHUVFLHQFH 1HZ
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722 6LOHU DQG )ULHG 0 f %LRRKHP 6WXOEHUJ 0 3 DQG 1RYHOOL f 0HWKRGV (Q]\PRO 7DNDJL 0 DULG 2JDWD f %LRRKHP %LRSK\V 5HV &RPPXQ 7DPDRNL 7 7KRPDV DQG 6FKLQGOHU f 1DWXUH 7DWD 3 DQG :LO LDPV$VKPDQ + f (XU %LRRKHP 7DWD 5 f %LRRKHP 7D\ORU 0 DQG 6FKLPNH 5 7 f %LRO &KHP 7UDXW 5 DQG 0XQUR 5 ( f 0RO %LRO A 8HQR\DPD DQG 2QR 7 f %LRFKLP %LRSK\V $FWD 8WVXQRPL\D 7 DQG 5RWK 6 f M &HOO %LRO 9DVVDOL 3 f 3URR 1DWO $FDG 6RL 86 YRQ (KUHQVWHLQ f 0HWKRGV (Q]\PRO $ :HOOHU ( 0 DQG 6FKHFKWPDQ $ 0 f 'HYHORS %LRO :LOOLV % DQG 6WDUU / f %LRO &KHP :LOVRQ 6 + DQG +RDJODQG 0 % f %LRRKHP =DFFKHR DQG *URVVL & ( f (PEU\RO H[S 0RUSK =DPHFQLN 3 & DQG .HOOHU ( % f %LRO &KHP =LPPHUPDQ + DQG )ULHG 0 f &RPS %LRRKHP 3K\VLRO %

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